Serine hydrolases (SHs) are one of the largest and most diverse enzyme families in the eukaryotic and prokaryotic proteomes, with a membership that includes lipases, esterases, thioesterases, peptidases/proteases, and amidases. The important biological roles played by SHs have led to the development of clinically approved drugs that target members of this enzyme family to treat diseases such as obesity, diabetes, microbial infections, and Alzheimer's disease. Despite these advances, most of the 200+ mammalian SHs remain poorly understood in terms of their biochemical and cellular activities. Pursuit of this knowledge would benefit from the development of selective inhibitors to probe the function of individual SHs in living systems. This constitutes an exciting, but challenging task that has been successfully accomplished for only a handful of SHs to date.
All SHs possess a serine nucleophile required for catalytic activity, opening up the opportunity to develop mechanism-based inhibitors that inactivate these enzymes by covalent modification. Among the classes of inhibitors that have been shown to react with the serine nucleophile of SHs, fluorophosphonates (FPs) and carbamates are exceptional in that they show negligible cross-reactivity with other nucleophilic enzymes such as cysteine hydrolases. FPs are highly reactive and provide broad, nearly complete coverage of the SH superfamily. This feature has promoted the use of reporter-tagged FPs for activity-based protein profiling (ABPP) investigations of SHs, but limits the utility of FPs as pharmacological probes for specific members of this enzyme class. Certain carbamate (R—O—C(O)—NR2) compounds, on the other hand, have been developed that show excellent selectivity for individual SHs. These inhibitors have proven to be valuable research tools and, in certain cases, advanced to the stage of approved drugs (e.g., rivastigmine, which targets acetylcholine esterase (ACHE) to treat Alzheimer's disease). Despite considerable screening efforts, however, efficacious and selective carbamate inhibitors have been identified for only a fraction of mammalian SHs, pointing to the need for alternative chemical classes of SH inhibitors. The present invention addresses these needs.
For example, biosynthesis of the endocannabinoid, 2-arachidonoylglcerol (2-AG) is enzymatically regulated by two distinct diacylglycerol lipase (DAGL) enzymes, DAGLA and DAGLB. In contrast with the enzymatic mechanisms regulating 2-AG degradation, relatively little is known about the DAGL enzymes with respect to their in vivo physiological functions. Biochemical studies performed in vitro have provided evidence that these transmembrane serine hydrolases, which share very little sequence homology with each other, can catalyze the sn-1 selective cleavage of arachidonate-containing diglycerides to form 2-AG. Recent genetic studies with DAGLA and DAGLB knockout mice have provided in vivo evidence that the chronic absence of these enzymes result in decreased 2-AG levels in central and peripheral tissues, respectively. To date, no selective inhibitors have been available for the DAGL enzymes and the most widely used compound, tetrahydrolipstatin (THL) has been shown to have potent activity against numerous other serine hydrolases in complex proteomes. In addition, this broad-spectrum lipase inhibitor shows poor bioavailability in vivo, a feature that allows its use as an anti-obesity drug by restricting activity to the gastrointestinal tract. The dearth of suitable DAGL inhibitors available for in vivo studies has prompted a search for novel chemotypes capable of inactivating these lipases in a selective manner. However, several challenges are associated with developing DAGL inhibitors, namely the lack of available assays required for medium-throughput to high-throughput screening. A significant challenge in the development of in vivo active DAGL inhibitors is the inability to measure the endogenous activity of these enzymes. In fact, no studies to date have shown that the endogenous enzymes are catalytically active in living cells or tissues, complicating interpretation of metabolic changes seen in genetic models.